Vinculin plays a pivotal role in cell adhesion and migration by providing the link between the actin cytoskeleton and the transmembrane receptors, integrin and cadherin. We used a combination of electron microscopy, computational docking, and biochemistry to provide an atomic model of how the vinculin tail binds actin filaments. The vinculin tail actin binding site comprises two distinct regions. One of these regions is exposed in the full-length autoinhibited conformation of vinculin, whereas the second site is sterically occluded by vinculin's N-terminal domain. The partial accessibility of the F-actin binding site in the autoinhibited full-length vinculin structure suggests that F-actin can act as part of a combinatorial input framework with other binding partners such as alpha-catenin or talin to induce vinculin head-tail dissociation, thus promoting vinculin activation. Furthermore, binding to F-actin potentiates a local rearrangement in the vinculin tail that in turn promotes vinculin dimerization and, hence, formation of actin bundles.

Arp2/3 complex initiates the growth of branched actin‐filament networks by inducing actin polymerization from the sides of pre‐existing filaments. Nucleation promoting factors (NPFs) are essential for the branching reaction through interactions with the Arp2/3 complex prior to branch formation. The modes by which NPFs bind Arp2/3 complex and associated conformational changes have remained elusive. Here, we used electron microscopy to determine three‐dimensional structures at ∼2 nm resolution of Arp2/3 complex with three different bound NPFs: N‐WASp, Scar‐VCA and cortactin. All of these structures adopt a conformation with the two actin‐related proteins in an actin‐filament‐like dimer and the NPF bound to the pointed end. Distance constraints derived by fluorescence resonance energy transfer independently verified the NPF location. Furthermore, all bound NPFs partially occlude the actin‐filament binding site, suggesting that additional local structural rearrangements are required in the pathway of Arp2/3 complex activation to allow branch formation.

Integrins are bidirectional, allosteric transmembrane receptors that play a central role in hemostasis and arterial thrombosis. Using cryo-electron microscopy, multireference single-particle reconstruction methods, and statistics-based computational fitting approaches, we determined three-dimensional structures of human integrin αIIbβ3 embedded in a lipid bilayer (nanodiscs) while bound to domains of the cytosolic regulator talin and to extracellular ligands. We also determined the conformations of integrin in solution by itself to localize the membrane and the talin-binding site. To our knowledge, our data provide unprecedented three-dimensional information about the conformational states of intact, full-length integrin within membrane bilayers under near-physiological conditions and in the presence of cytosolic activators and extracellular ligands. We show that αIIbβ3 integrins exist in a conformational equilibrium clustered around four main states. These conformations range from a compact bent nodule to two partially extended intermediate conformers and finally to a fully upright state. In the presence of nanodiscs and the two ligands, the equilibrium is significantly shifted toward the upright conformation. In this conformation, the receptor extends ∼20 nm upward from the membrane. There are no observable contacts between the two subunits other than those in the headpiece near the ligand-binding pocket, and the α- and β-subunits are well separated with their cytoplasmic tails ∼8 nm apart. Our results indicate that extension of the ectodomain is possible without separating the legs or extending the hybrid domain, and that the ligand-binding pocket is not occluded by the membrane in any conformations of the equilibrium. Further, they suggest that integrin activation may be influenced by equilibrium shifts.

A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40-50 (or 40-62 in the arginine limited digest) and residues 373-375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 A, constrains to that range the distance between the gamma-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin [Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44-49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836]. The effect of cross-linking on the function of actin is described in the companion papers.

No abstract

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degrees C the ability to activate myosin ATPase at a 100-fold slower rate and unfolded in CD melting experiments at a temperature higher by 11 degrees C than the un-cross-linked actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure but showed a change in the orientation of subdomain 2 and a decrease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major changes upon 50% and 90% ANP cross-linking of F-actin; the Kd and Km values were little affected by the cross-linking, and the Vmax decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decreased the mean speed and the number of sliding filaments in the in vitro motility assays by approximately 35% while the relative force, as measured by using external load in these assays, was inhibited by approximately 25%. The mean speed of actin filaments decreased with the increase in their cross-linking and approached 0 for the 90% cross-linked actin. Also examined were actin filaments reassembled from cross-linked and purified ANP cross-linked dimers, trimers, and oligomers. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behavior in the in vitro motility assays. Filaments made of cross-linked dimers moved at approximately 50% of the speed of the un-cross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely, and the oligomer-made filaments did not move at all. These results show the uncoupling between force generation and other events in actomyosin interactions and emphasize the role of actin filament structure and dynamics in the contractile process.

Residues 262-274 form a loop between subdomains 3 and 4 of actin. This loop may play an important role in actin filament formation and stabilization. To assess directly the behavior of this loop, we mutated Ser 265 of yeast actin to cysteine (S265C) and created another mutant (S265C/C374A) by changing Cys 374 of S265C actin to alanine. These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys 374 or Cys 265 . These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin. The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin. Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than wild-type (WT) actin, although the critical concentration was not affected. Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was ϳ50% less than the sum of the fluorescence of the individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins. An excimer peak was present in the emission spectrum of labeled S265C F-actin and in the labeled S265C/C374A-WT actin copolymers. These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 Å of one another. Finally, when bound to labeled S265C/ C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe. Both proteins had no effect on excimer fluorescence. These results help establish the orientation of monomers in Factin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.The crystal structure of the actin monomer has been elucidated as part of a 1:1 complex with three actin-binding proteins (1-3). However, the structure of the two-stranded actin filament at atomic resolution has not been determined due to the inability to date to crystallize F-actin. One F-actin model, proposed by Schutt et al. (2,4), is based on the profilin/␤-actin ribbon structure in which actin monomers contact each other in a continuous fashion, with profilin molecules bridging between the actins on the outside of the structure. In this structure, actin subdomains 1 and 2 are near the center of the ribbon, whereas the subdomain 3/4 interface is near its exterior. Schutt et al. have proposed that the ribbon can be transformed into a classical ADP-containing helical filament by a compression and a twist. However, coordinates of this filament model have not yet been published.Holmes et al. (5) have generated an alternative model based on fitting the coordinates of the monomer into a density map generated from low angle x...

No abstract